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C ampos G iménez : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014  1399

( e )  Sample dilution solvent .—Mix 90 mL mobile phase A with 10 mL mobile phase B. ( f )  Vitamin B 12 stock standard solution (100 µg/mL) .— Accurately weigh 20.0 mg vitamin B 12 into a 200 mL amber glass volumetric flask. Add about 150 mL water. Dissolve by sonication and stirring for a few minutes. Dilute to volume with water. Solution is stable for ≥6 months at –20°C. ( Note : Vitamin B 12 is sensitive to light. Conduct operations under subdued light, or use amber glassware. Keep all solutions away from direct light.) ( g )  Vitamin B 12 intermediate standard solution (400 ng/mL) .—Pipet 1 mL vitamin B 12 stock standard solution into a 250 mL amber glass volumetric flask. Make up to volume with water. ( h ) Vitamin B 12 working standard solutions for calibration (2, 10, 20, 40, 60, 100 ng/mL) .—Pipet into six separate 10 mL amber glass volumetric flasks, 50, 250, 500, 1000, 1500, and 2500 µL vitamin B 12 intermediate standard solution. Dilute to volume with sample dilution solvent, ( e ). E. Sample Preparation and Extraction ( a )  Sample reconstitution for powder samples .—Weigh 25.0 g sample into a 250 mL beaker. Add 200 g water at 40 ± 5°C. Mix with a glass rod until the suspension is homogeneous, or homogenize with a Polytron. Proceed as described in E ( d ) Extraction . ( b )  Sample reconstitution for amino acid-based products. — Weigh 25.0 g powder sample into a 250 mL beaker. Add 190 g water at 40 ± 5°C and 10 g skimmed milk powder. Mix with a glass rod until the suspension is homogeneous, or homogenize with a Polytron. In parallel, run a blank by replacing the sample by water. Dilute both, reconstituted sample and blank, twice in water (e.g., 50 g reconstituted sample or blank + 50 g water). Proceed as described in E ( d ) Extraction. ( c ) Sample preparation for liquid samples .—Mix well to ensure homogeneity of the sample portion. Proceed as described in E ( d ) Extraction . ( d )  Extraction .—Weigh 60.0 g sample suspension, E ( a ) and ( b ), blank, E ( b ), or liquid sample, E ( c ), into a 250 mL flat-bottom amber glass flask or Erlenmeyer with ground glass neck. Add 1 mL of 1% sodium cyanide solution, D ( b ). If the sample contains starch, add about 0.05 g α-amylase, mix thoroughly, stopper the flask, and incubate 15 min at 40 ± 5°C. Add 25 mL sodium acetate solution, D ( a ). Mix well. Place the flask in a boiling water bath for 30 min (or autoclave 30 min at 100°C). Cool the flask in an ice bath. Quantitatively transfer the content of flask to a 100 mL amber glass volumetric flask. Dilute to volume with water. Filter the solution through a folded paper filter. In the case of high-fat products, and if recovery is low, dilute the filtrate 1:3 in water before cleanup to improve recovery or repeat the extraction by using a smaller sample portion. ( e )  Immunoaffinity cleanup .—Let the immunoaffinity columns warm to room temperature by removing them from refrigeration at least 30 min before use. Place each immunoaffinity column on the rack. Open the caps and let the storage buffer drain by gravity. Close the lower cap. Load the column with 9 mL clear filtrate and close the upper cap. Place the column in a rotary shaker, and mix slowly for 10–15 min. Return the column to the support and let stand for

a few minutes. Open the caps to let the liquid drain by gravity. Wash the column with 10 mL water. With a syringe, insert about 40 mL air to dry the column. Elute with 3 mL methanol, and collect eluate in a 4 or 7 mL amber glass reaction vial. Rinse the column with 0.5 mL methanol, and with a syringe, insert about 20 mL air to collect all the methanol in the same vial. Evaporate the eluate at 50°C under a stream of nitrogen. Reconstitute the sample in 0.3 mL sample dilution solvent, D ( e ). Mix on a Vortex mixer. Transfer to a micro amber vial. F. Analysis ( a )  Chromatographic conditions .—Flow rate, 0.4 mL/min; injection volume, 50 µL; detection, UV at 361 nm; gradient elution, see Table 2014.02B . ( b )  Systemsuitability test .—Equilibrate the chromatographic system for at least 15 min. Inject a working standard solution three to six times, and check peak retention times and responses. Inject working standard solutions on a regular basis within a series of analyses. The coefficient of variation should not be higher than 2%. ( c )  Analysis .—Make single injections of standard and test solutions. Measure chromatographic peak response (height). ( d )  Identification .—Identify vitamin B 12 peak in the chromatograms of the test solution by comparison with the retention time and UV spectrum of the corresponding peak obtained for the standard solution. ( e )  Calibration .—Plot peak responses against concentrations (in ng/mL). Perform regression analysis. Calculate slope and intercept. Check the linearity of the calibration (R 2 > 0.99; standard error of calibration < 10%). ( f )  Quantitation .—Calculate the concentration of vitamin B 12 , in µg/100 g of product as reconstituted, as follows: ( − )× × × × × × where A= response (height) of the peak obtained for the sample solution, I = intercept of the calibration curve, S = slope of the calibration curve, V 0 = volume of the test solution (volume used to dissolve the test portion) in mL (100 mL), V 2 = volume in which the aliquot of sample solution is reconstituted after immunoaffinity cleanup (0.3mL), m=weight of the test portion, as reconstituted, in g (60 g), and V 1 = volume of the aliquot of sample solution loaded onto the affinity column (9 mL). For amino acid-based products calculate the vitamin B 12 content on the sample and on the blank, E ( e ); take into account the additional dilution factor 1/5 in the calculations. Deduct the amount of vitamin B 12 in the blank to the amount in the sample.

Table 2014.02B. Gradient elution Time, min

Mobile phase A, % Mobile phase B, %

0.0 1.7 2.5 2.9 3.9 4.0 8.0

90 90 75 10 10 90 90

10 10 25 90 90 10 10